In humans and in other species, the bone marrow contains both osteoblast and osteoclast precursor cells that can be made to differentiate into mature osteoblasts and osteoclasts, respectively. However, while osteoclast lineage cells have clearly been identified in the peripheral circulation, it remains controversial whether there is a comparable pool of circulating osteoblast lineage cells and what role, if any, these cells may play in normal or pathological bone remodeling. Previous studies have searched for circulating osteoblast lineage cells using traditional assays that involve culturing these cells on the basis of adherence to plastic. These studies have now demonstrated that osteoblast lineage cells are present in the circulation, but at extremely low concentrations. The present proposal is based on the assumption that osteoblast lineage cells have been difficult to find in the peripheral circulation in previous studies because these studies have used the criteria of plastic adherence to search for these cells. Thus, by their very nature, cells that adhere to plastic (or to other matrices) are unlikely to be present in significant amounts in the peripheral circulation. In contrast to previous studies, we are taking a different approach to search for circulating osteoblast lineage cells - namely, we are using flow cytometry to identify and collect cells expressing osteoblast-related proteins [osteocalcin (OC) or alkaline phosphatase (AP)] on their cell surface. In preliminary human studies, we have found that sorted AP positive cells express osteoblast-related genes (AP, OC, runx2/cbfal, osterix, and BMP-2), that OC positive cells are significantly increased in the circulation of adolescent boys around the time of expected peak growth velocity, and that (at least in a single subject) OC and AP positive cells are increased in the circulation of an adult male following a fracture. Based on these findings, the focus of this developmental R21 proposal is to provide a detailed characterization of the circulating OC and AP positive cells we have identified and a validation that our methods are detecting osteoblast lineage cells in the circulation that have the capacity to differentiate into mature osteoblasts. If successful, these studies would clearly meet one of the stated goals of this RFA, namely the "validation and determination of reliability of assays for various circulating progenitor populations in blood." The proposed validation studies would then set the stage for a broad range of clinical investigative studies examining changes in these circulating osteoblast lineage cells with aging, in osteoporosis, and following various therapeutic interventions.